This invention relates to bioassay techniques utilizing plant proteins expressed in a host cell system to identify their functionality in a plant G protein-coupled system, and is particularly useful in identifying plant G protein subunits and plant proteins that function in a manner similar to such G protein subunits and G protein coupled receptors. Other embodiments of the invention relate to host cells expressing such plant proteins, and especially plant G protein subunits, vectors useful for making such cells, and methods of making and using same.
In animal systems, the actions of many extracellular signals, for example: neurotransmitters, hormones, odorants and light, are mediated by receptors with seven transmembrane domains (G protein-coupled receptors) and heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins). G proteins are comprised of three subunits: a guanyl-nucleotide binding xcex1 subunit; a xcex2 subunit; and a xcex3 subunit [for review, see Conklin, B. R and Bourne, H. R. (1993) Cell 73, 631-641]. G proteins cycle between two forms, depending on whether GDP or GTP is bound to the xcex1 subunit. When GDP is bound, the G protein exists as a heterotrimer, the Gxcex1xcex2xcex3 complex. When GTP is bound, the xcex1 subunit disassociates, leaving a Gxcex2xcex3 complex. Importantly, when a Gxcex1xcex2xcex3 complex operatively associates with an activated G protein coupled receptor in a cell membrane, the rate of exchange of GTP for bound GDP is increased and, hence, the rate of disassociation of the bound Gxcex1 subunit from the Gxcex2xcex3 complex increases. The free Gxcex1 subunit and Gxcex2xcex3 complex are capable of transmitting a signal to downstream elements of a variety of signal transduction pathways. This fundamental scheme of events forms the basis for a multiplicity of different cell signaling phenomena. For additional review, see H. G. Dohlman, J. Thorner, M. Caron, and R. J. Lefkowitz, Ann. Rev. Biochem, 60, 653-688 (1991).
In plants, there is evidence that a number of plant signal transduction pathways, including red and blue light signaling (Warpeha et al., 1991; Romero and Lam, 1993; Neuhaus et al, 1993), K+ channel regulation of stomatal opening (Fairley-Grenot and Assmann, 1991; Li and Assmann, 1993; Armstrong and Blatt, 1995), and auxin signal transduction (Zaina et al., 1990) are regulated through G-protein intermediates. More specifically, in certain of these studies, red light-dependent responses in the tomato mutant aureus were measured after microinjection of either the GTP analogue GTPxcex3S or cholera toxin, either of which cause constitutive activation of a G-protein, and were found to produce the same effects as microinjecting phytochrome and exposing the plant to red light. Injection of inhibitors GDPxcex2S or pertussis toxin were effective in blocking red light-dependent responses.
It has also been suggested that an inhibitory G-protein modulates blue light-dependent K+ channel opening on the basis of electrophysiological studies using GTPxcex3S, GDPxcex2S and toxins. In separate experiments it was shown that blue light can activate a GTP-binding protein in the plasma membrane of peas. In rice, the binding of GTPxcex3S to vesicles in vitro is increased by auxin, while binding of GTPxcex3S decreased binding of auxin. This binding relationship suggests that auxin activation of a G-protein stimulates cell elongation. In addition, there is evidence for G-protein mediation of plant defense responses (Legendre et al., 1992, Vera-Estralla et al., 1994; Beffa et al., 1995).
Gxcex1-like proteins, having molecular weights close to that of the animal Gxcex1 subunits and recognized by antibodies against animal Gxcex1 subunits, have been detected in a large number of plant species (for reviews see Ma, 1994 and Kaufman, 1994) and three genes encoding G protein-xcex1 subunits have been identified. The first was cloned from Arabidopsis thaliana (Ma et al. 1990) using PCR primers based on sequences known to be conserved between animal G protein xcex1-subunits. The predicted protein has all of the consensus sequences for guanine nucleotide binding and hydrolysis that are characteristic of GTP-binding proteins and shows 36% identity with rat Gi(1-3) and bovine transducin. Using the Arabidopsis thaliana gene as a probe, genes were identified in both tomato (Ma et al., 1991) and soybean (Kim et al., 1995). The Arabidopsis thaliana tomato and soybean genes share over 80% identity, suggesting that plant G-protein xcex1-subunits may be highly conserved. Both Arabidopsis thaliana and tomato DNA appear to have single genes based on Southern blot analysis, whereas multiple genes may be present in soybean. Single genes encoding G protein xcex2 subunits have also been cloned from maize and Arabidopsis thaliana (Weiss et al., 1994). The predicted protein sequences shares 76% identity with each other and 41% identity with mammalian G protein xcex2 subunits.
Although the sequence of the plant G xcex1 subunits have been conserved relative to that of the mammalian G xcex1 subunits, there is no published data that demonstrates that the function of the plant protein is also conserved. Although physiological studies implicate G protein mediated responses in a number of pathways based on sensitivity of the response to cholera or pertussis toxins or the enhancement of the GTP binding to membranes by specific stimuli, this evidence is indirect. Prior to the work as detailed herein, the art has failed to provide direct evidence that these effects occur by the same mechanism by which they occur in other systems. In fact, it has recently been reported by one of the leaders in the field of plant G-protein molecular biology, that it is xe2x80x9c . . . unlikely that the plant G xcex1s are functional homologues of any of the non-plant ones.xe2x80x9d . . . (ibid. Hong Ma 1994).
Protein-mediated signaling systems are present in organisms as divergent as yeast, plant, and man. The yeast Saccharomyces cerevisiae is utilized as a model eukaryotic organism. Due to the ease with which one can manipulate the genetic constitution of the yeast Saccharomyces cerevisiae, researchers have developed a detailed understanding of many complex biological pathways. It has been demonstrated in numerous systems that the evolutionary conservation of protein structure is such that many heterologous proteins can substitute for their yeast equivalents. For example, mammalian Gxcex1 proteins can form heterotrimeric complexes with yeast Gxcex2xcex3 proteins [Kang, Y.-S., Kane, J., Kuijan, J., Stadel, J. M., and Tipper, D. J. (1990) Mol. Cell. Biol. 10, 2582-2590]. Screening assays utilizing yeast strains genetically modified to accommodate functional expression of plant G proteins offer significant advantages in research involving the identification of plant proteins that function in intracellular signaling systems, such as G-protein coupled systems.
A first aspect of the present invention is directed to a method of identifying a plant protein that functions in an intracellular signaling system such as a G protein coupled cellular signaling system. This is accomplished by providing a host cell with a nucleotide sequence encoding the plant protein it is desired to identify, which may be a plant protein suspected of being or functioning in a manner similar to a G protein subunit or a plant protein that is suspected of being or functioning in a manner similar to a G protein coupled receptor that functions in the intracellular signaling systems of mammals, insects (such as mammalian G protein coupled systems and insect G protein coupled systems), and the like. In certain preferred embodiments, an endogenous corollary component of a G protein coupled cellular signaling pathway, such as a Gxcex1 protein subunit, is rendered inoperative in the host cell. The host cell is transformed with a nucleotide sequence encoding the plant protein it is desired to identify and analyze, and the transformed host cell is allowed to grow under suitable conditions. The presence or absence, and sometimes the degree of said growth, is measured as an indication that the plant protein it is desired to identify has restored growth to said host cell, in the absence of its endogenous counterpart. From this information, it is determined that the target protein under study functions in a G-protein coupled cellular signaling system. In some cases, depending on the target under study, the protein may function in a xe2x80x9cG protein subunit-likexe2x80x9d manner, and particularly in the manner of a Gxcex1 subunit. G Protein components other than Gxcex1 may be rendered inoperative to identify and analyze various other components of G protein coupled systems, or intracellular systems that function as G-protein coupled systems. For example, the target protein may function as another G protein component such as a Gxcex2 or xcex3 component or a Gxcex2xcex3 complex. And in yet other preferred embodiments of the method of the invention, the plant protein it is desired to identify is suspected of being or functioning in a manner similar to a G protein coupled receptor. In such embodiments, the endogenous receptor or receptor-like protein is rendered inoperative or is removed entirely. The target protein of interest is introduced, and thus, in either event, the host cell is transformed with a nucleotide sequence that encodes a G-protein-coupled receptor-like protein.
In each of the above embodiments, all or a portion of the endogenous G protein signaling pathway may optimally be rendered inoperative and replaced with counterpart plant proteins, such as a Gxcex1 subunit, Gxcex2 subunit, and the like, or chimeric constructs containing a portion of the host cell derived subunit fused to a plant derived portion.
A second aspect of the present invention is directed to expression vectors and host cells transformed with the aforementioned target plant protein it is desired to identify. In certain preferred embodiments, the host cell contains a first heterologous nucleotide sequence which encodes a protein of interest, which is suspected of being a protein component that functions in an intracellular system, such as a G protein coupled cellular signaling system. Such nucleotide sequence may encode a G protein-coupled receptor protein, a G protein subunit, or a plant protein that is suspected of functioning in a manner analogous to a G protein coupled receptor or G protein subunit. In certain preferred embodiments, the nucleotide sequence encodes all or a portion of a G protein (such as an xcex1, xcex2, or xcex3 subunit), wherein the G subunit is plant-derived, at least in part. Various chimeric and trimeric hybrid G protein constructs are within the contemplation of the present invention. For example, in certain other embodiments, all or a portion of a nucleotide sequence encoding a heterologous plant G protein xcex1 subunit is fused to a nucleotide sequence from a yeast G protein or a mammalian G protein xcex1 subunit. Also within the contemplation of the expression vectors and host cells described herein are those transformed with a first heterologous nucleotide sequence which encodes a protein suspected of being a G protein coupled receptor-like protein and a second nucleotide sequence which encodes all or a portion of plant derived G protein subunit, as described above. In the most preferred embodiments, the expression system is yeast, and the expression vectors and transformed cells may usefully contain a third heterologous nucleotide sequence comprising a pheromone-responsive promoter and an indicator gene positioned downstream from the pheromone-responsive promoter and operatively associated therewith.
The vectors and cells utilizing the preferred yeast expression system may further contain several mutations effective to disconnect the pheromone responsive signal transduction pathway of the endogenous yeast host cell from that host cell""s natural cell cycle arrest pathway, or to otherwise increase sensitivity of the response of the cell to activation. In certain preferred embodiments, wherein a yeast cell is the host cell, these mutations include 1) a mutation of the yeast SCG1/GPA1 gene, which inactivates the yeast Gxcex1 protein, facilitating interaction of the heterologous plant protein with the G protein; 2) a mutation of a yeast gene to inactivate its function and enable the yeast cell to continue growing in spite of activation of the pheromone response signal transduction pathway, preferred embodiments being mutations of the FAR1 and/or FUS3 genes; and, 3) a mutation of a yeast gene, the effect of which is to greatly increase the sensitivity of the response of the cell to receptor-dependent activation of the pheromone response signal transduction pathway, preferred genes in this regard being the SST2, STE50, SGV1, STE2, STE3, PIK1, AFRI, MSG5, and SIG1 genes.
A third aspect of the present invention is chimeric expression constructs and host cells transformed therewith comprising a first nucleotide sequence encoding a plant protein suspected of functioning in a G protein coupled cellular signaling pathway, and further comprising in operative association therewith, a second nucleotide sequence which encodes an endogenous non-plant protein of the host cell or functional heterologous equivalent, which would be the corollary G protein component. Such constructs and cells may also contain a third heterologous nucleotide sequence comprising a pheromone-responsive promoter and an indicator gene positioned downstream from the pheromone-responsive promoter and operatively associated therewith. When the host cell is a yeast cell, the constructs and cells may further contain the mutations discussed above.
A productive signal is detected in a bioassay through coupling of the transformed heterologous protein to the host cell""s signal transduction pathway.
A fourth aspect of the present invention is a method of assaying compounds to determine effects of ligand binding to heterologous plant receptors by measuring effects on cell growth. In certain preferred embodiments, host cells are cultured in appropriate growth medium to cause expression of heterologous plant proteins, which become dispersed in a liquid medium or embedded in a solid phase medium, such as agar, and then exposed to substances applied to the surface of the vesicles or plates containing same. Effects on the growth of cells are expected around substances that activate the heterologous plant protein and the signal transduction pathway. Increased growth may be observed with substances that act as agonists, while decreased growth may be observed with those that act as antagonists.
The term xe2x80x9cG protein coupled cellular signaling systemxe2x80x9d as used herein refers to intracellular signaling systems that perform an intracellular signaling function similar to the well-known mammalian G protein-coupled receptor systems as discussed herein in the Background of the Invention, and display some of the structural hallmarks of such a system. The terms xe2x80x9cGxcex1-like, Gxcex2-like, Gxcex3-like, and Gxcex3xcex2-like complexxe2x80x9d inter alia, are intended to include mutants and homologs thereof and encompass proteins that function in a G protein coupled cellular signaling system, or equivalent intracellular signaling system in a manner analogous to the well-studied mammalian systems.
The term xe2x80x9cchimericxe2x80x9d as used herein generally refers to a protein expressed by a recombinant nucleotide sequence that is made by joining separate fragments of nucleotide sequences from more than one organism or species, or from more than one gene of the same organism or species. The term is used interchangably with fusion protein or construct and hybrid protein or construct and is not meant to limit such chimerics to those obtained solely through recombinant processes, it being understood that there may be other means of constructing same. xe2x80x9cTrimericxe2x80x9d refers specifically to a construct comprising three such separate protein portions.
The term xe2x80x9cheterologousxe2x80x9d as used herein with respect to a host cell and nucleotide sequence expression constructs and techniques, refers to nucleotide sequences, proteins, and other materials originating from organisms other than that particular host cell. Thus, mammalian, avian, amphibian, insect, plant, and yeast should all be considered heterologous to one another.
The term xe2x80x9cmammalianxe2x80x9d as used herein refers to any mammalian species (e.g. human, mouse, rat, and monkey).
The term xe2x80x9cnucleotide sequencexe2x80x9d is meant to include all forms of linear polymers comprising nucleotide bases, without limitation, including when appropriate, DNA, genomic DNA, cDNA, RNA, synthetic oligonucleotides, and the like.
The terms xe2x80x9creceptorxe2x80x9d or xe2x80x9creceptor-likexe2x80x9d as used herein are interchangeable and intended to encompass proteins that are identified as receptors or that function as a receptor and are further intended to include subtypes of proteins, and mutants and homologs hereof, along with the nucleotide sequences encoding same. One skilled in the art will also understand that in some instances, it may not be necessary that the entire receptor be expressed to achieve the purposes desired. Accordingly, the term receptor is meant to include truncated and other variant forms of a given receptor, without limitation.
The term xe2x80x9cupstreamxe2x80x9d and xe2x80x9cdownstreamxe2x80x9d are used herein to refer to the direction of transcription and translation, with a sequence being transcribed or translated prior to another sequence being referred to as xe2x80x9cupstreamxe2x80x9d of the latter.
In the method and constructs of the present invention, a host cell is transformed with a nucleotide sequence encoding a target plant protein it is desired to identify, characterize, and/or analyze. Any plant protein that is suspected of functioning in a G protein-coupled cellular signaling system or the like, such as a G protein coupled receptor, a protein that functions in a manner similar to G protein coupled receptors in intracelluar signaling, or portions thereof, as well as the plant G protein subunits themselves or proteins suspected of being such subunits or like components may be identified and assayed in accordance with the identification method and constructs of the present invention.
In certain embodiments of the methods of the invention, it is desirable to render inoperative all or a portion of the host cell""s endogenous G protein coupled cellular signaling pathway. This may be accomplished through any of a variety of means, such as by deletion of the endogenous gene suspected of being an integral component in such signaling pathway, disruption of such endogenous gene by insertion of a foreign DNA sequence, and expression of an antisense G protein gene, to name but a few techniques. In certain embodiments, the endogenous G protein-coupled pathway component that is rendered inoperative may be replaced with a corresponding plant G protein-coupled pathway component, or a hybrid construct containing all or a portion of a host cell""s endogenous component, or its functional equivalent from such heterologous system, fused to such plant protein. Any DNA sequence which codes for a plant G subunit may be used to prepare the constructs of the invention, or even a protein suspected of functioning as such a plant G protein subunit. Examples of such plant proteins include, but are not limited to, Arabidopsis thaliana G protein xcex1 subunit, Arabidopsis thaliana G protein xcex2 subunit, tomato G protein xcex1 subunit, soybean G protein xcex1 subunit, maize G protein xcex2 subunit, as further discussed in the Ma et al. (17-19) and Kim et al. (13) publications as listed on the attached Bibliography.
One skilled in the art will understand from the teachings as presented herein that the G proteins useful in the constructs and yeast cells of the present invention may comprise plant Gxcex1 subunits, or chimeric plant/mammalian Gxcex1 subunits, yeast/plant Gxcex1 subunits, or trimeric yeast/mammalian/plant versions. One can determine which configuration is best suited for adequate coupling to a cellular signaling system by simply constructing vectors as taught herein, transforming host cells, and determining the presence or absence of continued growth. Depending on the assay indicator system utilized, continued growth of said host cell is generally an indication that the G protein construct is functioning in said host cell in place of the functional endogenous G protein coupled cellular signaling complex. In certain preferred embodiments, the G protein xcex1 subunit is the plant subunit of choice, and especially that derived from Arabidopsis thaliana. 
Certain chimeric G protein constructs may also provide enhanced signal transduction with regard to particular heterologous receptors. One skilled in the art may prepare useful chimeric constructs by fusing operative regions of intracellular signaling components. Such operative regions may be selected in accordance with the teachings in this art. For example, the sites of interaction between Gxcex1 subunits and Gxcex2xcex3 subunit complexes or G protein-coupled receptors have been described (for review see B. R. Conklin and H. R. Bourne, 1993 (3)). The sites of interaction between Gxcex1 subunit complexes and the Gxcex2xcex3 subunits have been mapped to the amino terminus of the G protein xcex1 subunit, and possibly to the hxcex12 (xcex12 helix) and i1 (insert sequence 1) regions of the protein. The carboxy terminus of the G protein xcex1 subunit contains the region of the protein for which the strongest evidence for interaction with the G protein coupled receptor exists. Other regions that may contain contact points include the amino terminus and the G5 region (the G regions are peptide sequences that are conserved in all GTPases). A chimeric G protein alpha subunit with enhanced interaction with the yeast G protein xcex2xcex3 complex may thus be constructed by fusing the amino terminus of the yeast G protein xcex1 subunit containing these Gxcex2xcex3 contact sites with the carboxy terminus of the plant G protein xcex1 subunit.
Any DNA sequence which codes for a Gxcex2 subunit or a Gxcex3 subunit or other such subunits, or other components that interact with a plant Gxcex1 may be used to practice the present invention, including the host cell""s endogenous components. G proteins and subunits useful for practicing the present invention include subtypes, and mutants and homologs thereof, along with the nucleotide sequences encoding same. The host cells may express endogenous Gxcex2 and/or Gxcex3, a Gxcex2xcex3 complex, or appropriate plant counterpart subunits, or may optionally be engineered to express heterologous Gxcex2 and/or Gxcex3 (e.g., mammalian, plant, or various combinations thereof) in the same manner as they would be engineered to express heterologous Gxcex1 (see reference 22).
Heterologous nucleotide sequences are expressed in a host by means of an expression xe2x80x9cconstructxe2x80x9d or xe2x80x9cvector.xe2x80x9d An expression vector is a replicable nucleotide construct in which a nucleotide sequence encoding the protein of interest is operably linked to suitable control sequences capable of affecting the expression of a protein or protein subunit coded for by the nucleotide sequence in the intended host. Examples of cloning vector systems that may be utilized to obtain and/or amplify plasmids encoding the proteins it is desired to express include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA; plasmid DNA or cosmid DNA expression vectors. Host-expression systems useful for expression of the proteins it is desired to express include yeast carrying the recombinant yeast expression vectors containing the coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence or a nucleic acid containing the coding sequence; or animal cell systems infected with recombinant virus expression vectors (e.g., adenovirus, vaccinia virus) or containing stably integrated nucleotide sequences. Vectors useful for practicing the present invention include plasmids, viruses (including bacteriophage), and integratable DNA fragments (i.e., fragments integratable into the host genome by genetic recombination). The vector may replicate and function independently of the host genome, as in the case of a plasmid, or may integrate into the genome itself, as in the case of an integratable DNA fragment.
Generally, suitable control systems capable of affecting the expression of the protein of interest are selected in accordance with the vector/host system utilized, and are in operable association with the nucleotide sequence it is desired to express. Eukaryotic control sequences generally include a transcriptional promoter. However, it may also be appropriate that a sequence encoding suitable mRNA ribosomal binding sites be provided, and (optionally) sequences which control the termination of transcription. DNA regions are operably associated when they are functionally related to each other. For example: a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of leader sequences, contiguous and in reading frame.
In addition, a promoter operable in a host cell is one which binds the RNA polymerase of that cell, and a ribosomal binding site operable in a host cell is one which binds the endogenous ribosomes of that cell. Suitable vectors may in some instances also contain replicon control sequences (when the vector is non-integrating) which are derived from species compatible with the intended expression host.
The above-mentioned expression elements of these systems vary in their strength and specificities. Thus, depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, promoters such as pL of bacteriophage xcex, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used.
In cases where plant expression vectors are used, the expression of the coding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al., 1984, Science 224:838-843); or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, biolistics, microinjection, electroporation, etc. For reviews of such techniques see, for example, Weissbach and Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson and Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.
When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used; when generating cell lines that contain multiple copies of the DNA SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker. In cases where an adenovirus is used as an expression vector, the coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the protein in infected hosts. (E.g., See Logan and Shenk, 1984, Proc, Natl. Acad. Sci. (USA) 81:3655-3659). Alternatively, the vaccinia 7.5K promoter may be used. (E.g., see Mackett et al., 1982, Proc. Natl. Acad. Sci. (USA) 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931).
An alternative expression system which could be used to express the protein of interest is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of the coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (E.g., see Smith et al., 1983, J. Viol. 46:584; Smith, U.S. Pat. No. 4,215,051).
Specific initiation signals may also be required for efficient translation of inserted coding sequences. These signals include the ATG initiation codon and adjacent sequences. In some cases where the entire gene encoding the protein of interest, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. In other cases it may be necessary to modify these sequences to optimize the expression of a heterologous gene in a host system. However, in cases where only a portion of the coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bitter et al., 1987, Methods in Enzymol. 153:516-544).
Transformed host cells of the present invention are cells which have been transformed or transfected with the vectors constructed using recombinant DNA techniques or other suitable methodology, and express the protein or protein subunit coded for by the heterologous DNA sequences. The vectors and methods disclosed herein are suitable for use in host cells over a wide range of prokaryotic and eukaryotic organisms. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the nucleotide sequence it is desired to express and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques and in vivo recombination/genetic recombination (see Maniatis et al., 1989, Molecular Cloningxe2x80x94A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.). In the case of yeast cells, the vectors can also be introduced by mating cells with yeast of the opposite mating type that carry the vector. By utilizing techniques well known in the art in conjunction with the teachings as set forth herein, one skilled in the art may select the optimal host cell/vector expression system to express and identify the desired target plant proteins. In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, and the like.
Following the introduction of foreign DNA, engineered cells are allowed to grow under suitable growth conditions. For example, the cells may undergo a selective culturing protocol. The selectable marker in the recombinant plasmid confers resistance to the selection and allows transformed cells to grow to form colonies or foci, which in turn can be cloned and expanded into cell lines. The growth is measured. Growth generally indicates that the target plant protein functions in the intracellular signaling system while the absence of growth generally indicates that it does not. The degree of growth may also be assessed and extrapolated to the effectiveness of the target plant protein as an intracellular signaling component or to determine its function in the intracellular signaling machinery. This method may advantageously be used to engineer cell lines which express the plant protein, and which respond to mediated signal transduction, such as G protein coupled signaling systems. Detailed methodologies for selective culturing protocols suitable for use with a variety host cell expression systems may be found in Current Protocols in Molecular Biology [4].
An example of the numerous selection systems that may be used, include but are not limited to, the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanhine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can be employed in tk, hgprt or aprt cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O""Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147) genes. Recently, additional selectable marker genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine ([Hartman and Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047); and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.).
Recombinant yeast expression systems are particularly useful in the practice of the present invention, and are therefore preferred for use herein. A variety of yeast cultures, and suitable expression vectors for transforming yeast cells, are known. See e.g., U.S. Pat. Nos. 4,745,057; 4,797,359; 4,615,974; 4,880,734; 4,711,844; and 4,865,989. Saccharomyces cerevisiae is the most commonly used among the yeasts, although a number of other yeast species are commonly available, such as Schizosaccharomyces pombe, and the like. See. also, U.S. Pat. No. 4,806,472 (Kluveromyces lactis and expression vectors therefore); 4,855,231 (Pichia pastoris and expression vectors therefore). In certain embodiments, yeast vectors may contain an origin of replication from the endogenous 2 micron yeast plasmid or an autonomously replicating sequence (ARS) which confers on the plasmid the ability to replicate at high copy number in the yeast cell, centromeric (CEN) sequences which limit the ability of the plasmid to replicate at only low copy number in the yeast cell, a promoter, DNA encoding the heterologous DNA sequences, sequences for polyadenylation and transcription termination, and a selectable marker gene. Exemplary plasmids and detailed materials and methods for making and using same are provided in the EXAMPLES section.
Any promoter capable of functioning in yeast systems may be selected for use in the preferred expression constructs and host cells of the present invention. Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (PGK) [Hitzeman et al., (1980) J. Biol. Chem. 255, 2073] or other glycolytic enzymes [(Hess et al., (1968) J. Adv. Enzyme Reg. 7, 149; and Holland et al., (1978) Biochemistry 17, 4900], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate, decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657. Other promoters, which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase, 1,2,-isocytochrome C, acid phosphates, degradative enzymes associated with nitrogen metabolism, and the aforementioned metallothionein and glyceraldehyde-3-phosphate dehydrogenase, as well as enzymes responsible for maltose and galactose utilization, such as the galactose inducible promoter, GAL1. Particularly preferred for use herein are the PGK, GAL1, and alcohol dehydrogenase (ADH) promoters. Finally, in constructing suitable expression plasmids, the tennination sequences associated with these genes may also be ligated into the expression vector 3xe2x80x2 of the heterologous coding sequences to provide polyadenylation and termination of the mRNA. In preparing the preferred expression vectors of the present invention, translational initiation sites are chosen to confer the most efficient expression of a given nucleic acid sequence in the yeast cell [see Cigan, M. and T. F. Donahue 1987, GENE, Volume 59, pp. 1-18, for a description of suitable translational initiation sites]. A particularly preferred nucleotide expression vector useful for carrying out the present invention comprises such an aforementioned promoter sequence, positioned upstream to the translational initiation site of the heterologous nucleotide sequence encoding for the plant protein it is desired to express. Particularly preferred promoters in this regard are the GAL1, PGK, and ADH promoters.
Any of a variety of means for detecting the effects of the transduced plant protein on a G protein coupled cellular signaling system of the host cell may be utilized. For example, measurement of the disassociation of Gxcex1 from Gxcex2xcex3 can be made through conventional mechanical disruption techniques. However, detectable biological responses may also lend themselves to measurement. One such biological response is the activation of the pheromone induced mating signal transduction pathway, which is the preferred method of detecting the effects in the assay systems herein presented, the basic premise of which is discussed in more detail in PCT 95/21925. As set forth therein, selected mutations in endogenous yeast genes can lead to hypersensitivity to pheromone and an inability to adapt to the presence of pheromone. For example, introduction of mutations that interfere with function into yeast strains expressing the target proteins enables the development of extremely sensitive bioassays for the effect of the target protein, or for the effect of substances that interact with G protein coupled receptors. Other mutations e.g. STE50, sgvl, ste2, ste3, pik1, msg5, sig1, and afr1, have the similar effect of increasing the sensitivity of the bioassay. One skilled in the art will understand that increased sensitivity of the assay systems is attained through a variety of mutations, such as deletion of one or more of these aforementioned genes, introduction of nucleotide substitutions that disrupt the activity of the protein, introduction of mutations that down-regulate their expression, or in certain instances, effecting their overexpression. For example, in the STE50 construct, overexpression of the gene is desired, not deletion of the gene. Thus, introduction of a constellation of mutations in the mating signal transduction pathway results in a yeast cell well suited to expression of plant proteins, which are able to functionally respond in an intracellular signaling system.
In conjunction with one or more of the above-referenced mutations, a particularly convenient method for detecting the effects of the plant protein on the cellular signaling system is to utilize a conventional genetic indicator system. Thus, in certain preferred embodiments, the cells are provided with an additional heterologous nucleotide sequence, comprising a pheromone-responsive promoter and an indicator gene positioned downstream from the pheromone-responsive promoter and operatively associated therewith. With such a sequence in place, the detecting step can be carried out by monitoring the expression of the indicator gene in the cell. Any of a variety of pheromone responsive promoters could be used, examples being promoters driving any of the aforementioned pheromone responsive genes (e.g. mFxcex11, mFxcex12, MFA1, MFA2, STE6, STE13), the BAR1 gene promoter, and the FUS1 gene promoter. Likewise, any of a broad variety of indicator genes could be used, with examples including the HIS3, G418r, URA3, LYS2, CAN1, CYH2, and LacZ genes. A particularly preferred reporter gene construct is utilized by fusing transcription control elements of a FUS1 gene to HIS3 protein coding sequences, and replacing the original FUS1 gene with this reporter construct. Expression of the HIS3 gene product is thereby placed under the control of the pheromone signal transduction pathway. Yeast strains (his3) bearing this construct are able to grow poorly on supplemented minimal medium lacking histidine, and are sensitive to an inhibitor of the HIS3 gene product. Activation of gene expression results in increased growth on this medium. In other preferred embodiments, plasmids carry a FUS1-lacZ gene fusion. Expression of these gene fusions is stimulated in response to receptor activation by binding of pheromone. Therefore, signal transduction can be quantitated by measuring xcex2-galactosidase activity generated from the FUS1-lacZ reporter gene, or detection of enhanced growth on minimal media in yeast expressing the FUS1-HIS3 reporter gene.
Other useful reporter gene constructs, still under the control of elements of the pheromone signal transduction pathway, but alternative to the above-discussed reporter systems, may involve signals transduced through other heterologous effector proteins that are coexpressed. For example, 1) stimulation of a heterologous adenylylcyclase may permit a yeast strain lacking its own adenylylcyclase due to mutation in the cdc35 gene to survive, 2) stimulation of a heterologous G protein-coupled potassium channel may permit a yeast strain unable to grow in medium containing low potassium concentration [(trk1, trk2), for example, see Anderson, J. A. et al (1992)( Proc. Natl. Adad. Sci. USA 89, 3736-3740] to survive, or 3) stimulation of a heterologous PLC-xcex2 may permit a yeast strain lacking its own PLC [(plc)], for example, see Payne, W. E. and Fitzgerald-Hayes, M. (1993) Mol. Cell Biol. 13,4351-4363] to survive.
Any DNA sequence which codes for an adenylylcyclase may be used to practice the present invention. Examples of adenylylcyclase include the product of the D. melanogaster Rutabaga gene and the mammalian subunit types I-VIII [for review see, Tang, W. -J. and Gilman, A. G. (1992) Cell 70, 869-872], and mutants and homologs thereof, along with the DNA sequences encoding same, which are useful for practicing the present invention.
Any DNA sequence which codes for a G protein-gated potassium channel may be used to practice the present invention. Examples of G protein-coupled potassium channel include GIRK1 [Kubo, Y. Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y. (1992) Nature 365, 802-806], subunits useful for practicing the present invention, and mutants and homologs thereof, along with the DNA sequences encoding same.
Any DNA sequence which codes for a phospholipase protein may be used to practice the present invention. Examples of phospholipase proteins include the D. melanogaster norpA gene product and the PLC-xcex2 proteins [for review, see Rhee, S. G., and Choi, K. D. (1992) J. Biol. Chem. 267, 12392-12396], subunits useful for practicing the present invention, and mutants and homologs thereof, along with the DNA sequences encoding same.
Transformed host cells of the present invention express the proteins or protein subunits coded for by the heterologous DNA sequences. When expressed, the plant protein is capable of functional interaction with the other G proteins of the intracellular signaling system. The G protein coupled receptors and some effectors, such as adenylyl cyclases and ion channels, are integral membrane proteins. The plant protein may also be associated with the membrane, as is the case with G proteins or the effector cGMP phosphodiesterase, or may be a membrane lipid, such as the phosophatidylinositol-phopholipases. Some of the G protein in the cell may be concentrated on intracellular membranes including the endoplasmic reticulum, golgi complex, endosomes and secretory vesicles (F. A. Barr et al., 1992).
Implementation of the methods and constructs described herein will facilitate description of structural and functional aspects of receptor-ligand and receptor-G protein interactions. The role of plant proteins that modify the response of receptors and G proteins may be worked out in detail with the assistance of this powerful genetic system. Importantly, the system provides a generalized approach to the study of the functioning and identification of components of the G protein coupled transduction system in plants, as well as a generalized approach to screening assays utilizing the G protein coupled signal transduction system. Once the plant intracellular signaling system is better understood, any of a variety of pesticides, herbicides, fungicides and the like may be designed, having novel mechanisms of action. The present invention provides expression constructs and assay systems adapted to receive any of a variety of plant proteins it is desired to identify, in the form of xe2x80x9cexpression cassettesxe2x80x9d. The plant protein it is desired to study is simply inserted into the vectors herein provided, and expressed in appropriate host cells. The systems presented herein also facilitate the identification of ligands for plant G protein-coupled proteins. In certain assay embodiments of the invention, any of a variety of substances may be contacted with a host cell expressing the target plant protein, to ascertain the effect of such substance on the intracellular signaling system. Such substances may comprise organic compounds, metal ions, peptides, proteins and the like, depending on the needs of the user. Suitable assay parameters, such as length of interaction, media utilized, and other test conditions are constructed in accordance with techniques generally utilized in the art.